SELECTION OF MULTIPLE DISRUPTION EVENTS IN ASPERGILLUS-FUMIGATUS USING THE OROTIDINE-5'-DECARBOXYLASE GENE, PYRG, AS A UNIQUE TRANSFORMATION MARKER

A 8.6-kb disruption cassette, referred to here as a pyrG-blaster and c onsisting of the Aspergillus niger pyrG gene flanked by a direct repea t that encodes the neomycin phosphotransferase of transposon Tn5 was c onstructed. Following transformation of a uridine/uracil auxotrophic p yrG strain of A. fumigatus, genomic insertions of the pyrG-blaster wer e obtained either by targeted gene replacement at the rodA locus, resu lting in the formation of hydrophilic spores, or by ectopic integratio n. In both cases, recombination between the two elements of the direct repeat could be selected in the presence of 5-fluoro-orotic acid and resulted in the excision of the A. niger pyrG gene, producing A. fumig atus uridine/uracil auxotrophs that retained their additional mutant p henotype because of the persistence of one of the two elements of the direct repeat at the site of insertion of the pyrG-blaster. Selection for uracil/uridine prototrophy can therefore be used again to disrupt another gene.